发酵液中他克莫司含量的测定方法研究

Studies on quantitative determination methods of tacrolimus in the fermentation broth

目的:他克莫司是一种新型免疫抑制剂,目前国内外报道的检测发酵液中他克莫司的方法还不多。本文建立定量检测发酵液中他克莫司的2种方法。方法:高效液相色谱法,确定流动相条件为0.1%磷酸溶液-乙腈(32:68),流速为1mL·min^(-1),检测波长为205nm,柱温为50℃;生物效价测定法,利用白色念珠菌为鉴定菌,采用类似于测定抗生素生物效价的杯碟法准确测定出了发酵液中他克莫司的含量。结果:高效液相色谱法测定结果表明在10～100μg·mL^(-1)的范围内他克莫司浓度与峰面积呈现良好的线性关系,平均回收率为99.94%(RSD=1.6%)。他克莫司对白色念珠菌有明显抑菌效果,生物效价测定法回归方程 Y=0.1285X-1.8936,r=0.9982,基本达到实验要求。结论:生物效价测定法简便、经济、快捷。将2种方法进行比较,证实生物效价测定方法准确、有效。

Objective： Tacrolimus is a novel immunosuppressant, and presently the measures to analyzed the fermentation titer of tacrolimus are still not many. This article established two methods to detect tacrolimus in the fermentation broth. Method： Determination of HPLC ： Determined the analysis method that the mobile phase was 0. 1% phosphoric acid -acetonitrile （32：68）, the velocity of flow was 1 mL ·min^- 1, and product peak was detected in the 205 nm and at 50 %;. Bioassay （estimation of biological potency） ：Used Candida albicans as identified bacterium and determined tacrolimus with bioassay as antibiotics. Results：The result with HPLC was that the concentration of tacrolimus and peak area appeared excellent linearity over the range of 10 -100 μg · mL^-1 ,and average recovery was 99.94% （ RSD = 1.6% ）. Also, we found tacrolimus could inhibit Candida albican, growth strongly, and set out production of tacrolimus in the fermentation broth by bioassay, with the regresstion equation was Y = 0. 1285X - 1. 8936 and coefficient was 0. 9982. Conclusion ：This bioassay method is simple, rapid, economical, and credible by comparison with HPLC.